Abstract The goal of the current Phase I SBIR project is to develop commercially scalable and reproducible in vitro 3-dimensional organotypic models of rat tissues including airway (bronchial and alveolar), intestine and liver. These models will replicate biological responses of the corresponding in vivo rat tissues and provide an alternative to traditional in vivo rat toxicity testing. The species (rat) and organ tissues (lung, liver and intestine) were chosen as initial models for commercial development in the current project based on specific stated interests of the funding agency and requests of prospective pharmaceutical industry customers. These rat- derived in vitro models will provide in vitro to in vivo translational information needed to predict toxicity of chemical and drug candidates by allowing for comparisons with existing in vivo rat data. These models will also enable comparisons with existing human cell-based in vitro organotypic models that use similar technologies. The translational information provided by these models will ultimately improve confidence and facilitate use of in vitro models for predicting human clinical outcomes. The Specific Aims of the Phase I project are as follows. Aim 1: Develop organotypic rat bronchial and alveolar airway epithelial models. AIM 2: Develop organotypic rat Intestine epithelial models. The airway and intestine models will be cultured on microporous membrane inserts at the air-liquid interface. AIM 3: Develop organotypic rat liver models: These will consist of hepatocytes and hepatic stellate cells co-cultured as spheroids. AIM 4: Demonstrate the interaction of intestine/liver and airway/liver models in pilot experiments using a perfused multi- organ chip (MOC). Troglitazone, an anti-diabetes drug that was withdrawn from the markets due to severe liver injury, will be used as a model drug to evaluate liver toxicity following delivery via the intestinal (oral) or alveolar (inhaled) epithelium. In Phase II, these models will be further evaluated in microphysiological MOC experiments to validate more complex multi-organ interactions and toxicological effects. Addition of immune components (macrophages, dendritic cells, Kupfer cells) will also be undertake during the Phase II period. Specific endpoints to be evaluated in the current work include tissue viability assessments, barrier disturbance, drug transport and/or metabolism, fibrotic responses, and innate immune responses. The results of the in vitro experiments conducted in the current project will be compared to existing historical data previously reported for in vivo animal studies and human clinical data (e.g. plasma concentrations following oral absorption, and known toxicological effects of drugs).